Apparatus and method for micron and submicron particle formation

ABSTRACT

An apparatus is described for micron and submicron particles formation of a substance using the GAS process, comprising a particles formation vessel ( 22 ) and means for introducing a solution of the substance and a supercritical fluid into the particles formation vessel ( 22 ), wherein said means comprise a nozzle ( 27 ) having a central orifice ( 39 ) serving to carry a flow of solution, and a plurality of separate outer orifices ( 41 ) serving to carry a flow of pure supercritical fluid or a flow of supercritical fluid mixed with a modifier, such that the solvent is extracted from the solution by the supercritical fluid and precipitation of micron and submicron particles occurs. Also a process is described, carried out with such an apparatus.

FIELD OF THE INVENTION

The present invention relates to an apparatus and method of forming veryfine particles of chemical compounds using fluid antisolventprecipitation. More particularly but not exclusively it relates to amethod of forming micro particles of proteins, for example proteins ofpharmaceutical interest.

BACKGROUND OF THE INVENTION

A large number of industries are interested in the production of micronand submicron particles for different applications. The need for anapparatus and a method to produce submicron particles is particularlypronounced in the pharmaceutical field.

There are several reasons for employing drugs as fine powders inpharmaceutics, such as the need to improve the bioavailability or therequirements for specific pharmaceutical forms (nasal, ophthalmic,injectables, modified release), etc.

The conventional techniques for particle size reduction (grinding,milling, spray drying, freeze drying) present many disadvantages, inparticular for biological active principles. For instance during theinitial step of freeze drying, the drug (protein) and the buffer andother ingredients tend to concentrate leading to changes in pH and ionicstrength; this can cause protein denaturation. Concerning spray drying,the main limitations of this technique are essentially high costs,thermal degradation and low efficiency with low yield and high levels ofresidual moisture.

Within the last decade, different processes have been proposed formicron and submicron particles formation by utilizing supercriticalfluid techniques (RESS, GAS, SEDS, PGSS (Precipitation from GasSaturated Solution)).

These processes have received considerable attention, because they allowhomogeneous particles with a diameter smaller than 1 micron to beobtained. In addition these processes allow very good control of sizeand morphology of powders, the compounds are not subject to mechanicaland thermal shock, and the powders obtained are free of any solvent.

Two processes for obtaining micro-particles by supercritical fluids haveattained high interest: Rapid Expansion of Supercritical Solutions(RESS) process (Tom, J. W., Debenedetti, P. G. “The formation ofbioerodible polymeric microsphere and micro particles by rapid expansionof supercritical solutions” BioTechnol. Prog. 1991, 7, 403–411.) and GasAnti-Solvent recrystallization (GAS) process (Gallagher, P. M., Coffey,M. P., Krukonis, V. J., Klasutis, N., Am. Chem. Symp. Ser., 1989, No.406).

In the RESS process the substance of interest is solubilized in asupercritical fluid and the solution is sprayed into a particleformation vessel through a nozzle: rapid expansion of the supercriticalsolution causes the precipitation of the solute. In some applications itis possible to add a subcritical solvent (modifier) to the supercriticalfluid.

A drawback of this technique is that only a few compounds are solubleenough in supercritical fluids, even if a modifier is used. In additionthe rapid expansion of supercritical solution through the nozzle cancause the freezing of supercritical fluid and the blockage of thenozzle.

In the GAS process a solute of interest is dissolved in a liquid solventthat is miscible with supercritical fluid, while the solute is notsoluble in the supercritical fluid.

The solution is sprayed through a nozzle into a particle formationvessel which is pressurized with supercritical fluid. The rapid andintimate contact between solution and supercritical fluid causes theextraction of solvent from solution in the supercritical fluid and leadsto the precipitation of solute as micro-particles. It is possible toenhance the solubility of the liquid solvent in the supercritical fluidby using a modifier. The GAS process overcomes the drawbacks of the RESSprocess and allows a better control of process parameters.

The crucial step of the GAS process is the mixing of solution andsupercritical fluid: in order to obtain an intimate and rapid mixing adispersion of solution as small droplets into the supercritical fluid isrequired. Different devices have been proposed to inject solution andsupercritical fluid into particle formation vessel in order to obtain agood mixing.

A simple capillary nozzle with a diameter between 0.1 and 0.2 mm hasbeen used first (Dixon D. J. and Johnston K. P., Formation ofmicroporous polymer fibers and oriented fibrils by precipitation with acompressed fluid antisolvent, J. App. Polymer Sci., 50, 1929–1942,1993).

This device shows high pressure drop along its length leading to a poorconversion of pressure into kinetic energy at the capillary outlet.

Debenedetti P. G., Lim G. B., Prud'Homme R. K. (U.S. Pat. No.006,063,910, May 16, 2000) use the GAS process to form protein microparticles. In this case the protein solution is sprayed through a laserdrilled platinum disc with a diameter of 20 micron and a length of 240micron inside the particles formation vessel containing thesupercritical fluid which is introduced by a different inlet. Thelaser-drilled platinum disc has an outside diameter of 3 mm, a thickness0.24 mm, and the orifice is 20 micrometers in diameter. This techniquehas been used to form particles of catalase and insulin (0.01% w/v) fromethanol/water (9:1 v/v) solutions using carbon dioxide as supercriticalfluid. The experiments were carried out at 8.8 MPa and 35° C.;supercritical fluid flow rate was about 36 g/min and the solution flowrate was about 0.35 cc/min.

Compared to a capillary nozzle, the laser drilled disc presents one mainadvantage: the ratio between length and diameter of the orifice allowsminimizing of the pressure drop and energy pressure is almost completelyconverted into kinetic energy; in such a way, very high solution ratesand very small droplets can be obtained.

In this process the supercritical fluid inlet is not optimized: thesolution injection occurs in an almost static atmosphere ofsupercritical fluid, with low turbulence.

Subramaniam B., Saim S., Rajewskj R. A., Stella V. (Methods for particlemicronization and nanonization by recrystallization from organicsolutions sprayed into a compressed antisolvent. U.S. Pat. No.5,874,029, Feb. 23, 1999) disclose use of a commercial coaxialconvergent-divergent nozzle to inject solution into a particle formationvessel. The nozzle has a convergent-divergent passage for the gasexpansion and an inner coaxial capillary tube. The solution injectedthrough the coaxial capillary tube is energized by the expanding gas.The gas that expands in the convergent-divergent nozzle can reachsupersonic velocities.

The transition from subsonic to supersonic rate in the nozzle leads tothe formation of a Mach disc which enhances dispersion of the solutionand mixing between solution and supercritical fluid. Subramaniam et al.propose as energizing gas an inert gas as helium or the supercriticalfluid. In the cited examples the authors use the supercritical fluid asthe energizing gas.

Even if to reach supersonic velocities very high pressure drops of theenergizing gas are required (about 40 MPa), the inventors operate atmilder conditions, using pressure drops of about 40 bar (4 MPa), so theycould not reach supersonic velocities. Notwithstanding, they claimsubstantial improvements compared to conventional GAS process.

Experimentally they recrystallised hydrocortisone and camptothecinobtaining powders in the range of nanoparticles (0.5–1 μm).

An advantage of this technique is that the supercritical fluid improvesthe solution spraying in order to obtain very fine droplets; anotheradvantage is due to the intimate mixing between solution andsupercritical fluid which occurs in a very small tract, at the nozzleoutlet.

The disadvantage of this technique is that the mixing between solutionand supercritical fluid occurs before entering into the particleformation vessel: this situation could lead to particle formation beforefluids enter into the particles formation vessel and consequentlyblockage of the nozzle.

Hanna M., York P. (WO patent application No 96/00610, Jan. 11, 1996)propose a new method and a new apparatus to obtain very small particlesby supercritical fluid technique named SEDS (Solution EnhancedDispersion by Supercritical Solution).

The process is based on a new coaxial nozzle: the solution expandsthrough an inner capillary with a diameter of 0.25 mm; the supercriticalfluid expands through an external coaxial pathway with a conicallytapering end; the diameter of conical zone at the end is about 0.2 mm.The mixing between the supercritical fluid and the solution occurs inthe conical zone. They also propose the use of a three ways nozzle: inthe added way a modifier can be fed in order to improve the mixing. Theyapply the SEDS technology for precipitation of small particles of watersoluble compounds, namely sugars (Lactose, Maltose, Trehalose andSucrose) and proteins (R-TEM beta-lactamase).

The modifier (methanol or ethanol) is introduced into the particlesformation vessel either together with the solution or, through adifferent inlet.

This nozzle allows a good and intimate mixing between the supercriticalfluid and the solution: the first contact between supercritical fluidand solution occurs in the conical shaped end, the two fluids emergefrom the nozzle outlet at high velocity and the supercritical fluidenergizes the liquid solution which breaks into small droplets in theparticles formation vessel.

The disadvantage of this technique is related to the contact betweensupercritical fluid and solution before entering into the particlesformation vessel; precipitation of the powder could occur in the nozzleand can eventually cause nozzle blockage.

The supercritical fluid velocity at the nozzle outlet is limited by theorifice diameter that is quite large.

It is known from GM-A-2 322 326 to provide modified apparatus forparticle formation using the SEDS technique. The apparatus comprises aparticle formation vessel and means for introducing a solution of thesubstance and a supercritical fluid into said particle formation vessel,said means comprising a nozzle having respective passages for thesolution and the supercritical fluid and separate outlets at downstreamends of the respective passages, such that in use contact between thesolution and the supercritical fluid first occurs in the particleformation vessel downstream of the separate outlets.

STATEMENTS OF INVENTION

The term “supercritical fluid” means a fluid at or above its criticalpressure and its critical temperature.

The term “solvent” means a liquid, which is able to form a solution withthe substance.

The term “substance” means a solid of pharmaceutical interest which issoluble in the solvent and which is substantially insoluble in thesrupercritical fluid.

The term “modifier” means a chemical which enhances solubility of thesolvent in the supercritical fluid.

An object of the present invention is to overcome the drawbacks, of theprior art techniques described above.

In particular, it is an object of the present invention to provide aprocess to obtain fine powders of a substance and an apparatus to makean intimate mixture of substance solution with the supercritical fluid.

Viewed from one aspect the invention provides apparatus for micron andsubmicron particle formation of a substance using the gas anti-solventrecrystallization (GAS) process, comprising a particle formation vesseland means for introducing a solution of the substance and asupercritical fluid into said particle formation vessel, said meanscomprising a nozzle having respective passages for the solution and thesupercritical fluid and separate outlets at downstream ends of therespective passages, such that in use contact between the solution andthe supercritical fluid first occurs in the particle formation vesseldownstream of the separate outlets, wherein the passages a wide diameterupstream portion which feeds a narrow diameter downstream portion.

Viewed from another aspect the invention provides a nozzle for theintroduction of a solution of a substance and a supercritical fluid in aparticle formation vessel for micron and submicron particle formation ofsaid substance using the gas anti-solvent recrystallization process, thenozzle comprising respective passages for the solution and thesupercritical fluid and separate outlets at downstream ends of therespective passages, such that in use contact between the solution andthe supercritical fluid first occurs downstream of the separate outlets,wherein the passages comprise a wide diameter upstream portion whichfeeds a narrow diameter downstream portion.

Viewed from a further aspect the invention provides a process for micronand submicron particle formation of a substance using the gasanti-solvent recrystallization. (GAS) process, comprising the feeding ofa supercritical fluid, pure or mixed with a modifier, and of a solution,through a nozzle, into a particle formation vessel at controlledpressure and temperature, such that the solvent is extracted fromsolution by the supercritical fluid and precipitation of micron andsubmicron particles occurs, wherein the supercritical fluid and is thesolution are fed through respective passages of the nozzle to exittherefrom via separate outlets at downstream ends of the respectivepassages, with contact between the supercritical fluid and the solutionfirst occurring in the particle formation vessel downstream of theseparate outlets, and wherein the passages comprise a wide diameterupstream portion which feeds a narrow diameter downstream portion.

The process according to the invention includes the co-introduction intoa particle formation vessel of a solution or suspension of the substancein a solvent, of a supercritical fluid and, preferably, of a modifier.The modifier is a compound which is soluble in the solvent and in thesupercritical fluid. The modifier is used when the solvent issubstantially insoluble with the supercritical fluid, or of lowsolubility.

When the solubility of the solvent in the supercritical fluid is low,the use of a modifier allows a better mixing between solution andsupercritical fluid.

When a modifier is used, the ratio of modifier flow rate and of solutionflow rate has to be chosen so as to have a high increase of solubilityof solvent in the supercritical fluid. The modifier can be introducedwith the supercritical fluid, with the solution or in part with thesupercritical fluid and in part with the solution; the way ofintroduction of the modifier greatly influences the extraction of thesolvent and the structure of particles that are formed.

For the precipitation of powders from aqueous solution using carbondioxide as supercritical solvent and ethanol as modifier the ratiobetween supercritical fluid flow rate and the modifier flow rate isabout 7, while the ratio between modifier flow rate and the solutionflow rate is about 20.

Thus, in one case the substance solution and a mixture of supercriticalfluid and modifier are separately introduced into the particle formationvessel. The modifier and the supercritical fluid are mixed before theintroduction into the particle formation vessel. Alternatively, themodifier may be mixed with the solution before introduction. In anotherversion of the process the modifier is introduced into the particleformation vessel in part with the solution and in part with thesupercritical fluid.

If the solvent is miscible with the supercritical fluid, the solution ofthe substance in the solvent and the supercritical fluid are separatelyintroduced into the particle formation vessel, in which mixing of thesupercritical fluid with the solution and extraction of the solvent bythe supercritical fluid occur.

The substance is preferably a pharmaceutical compound soluble in thesolvent and in the modifier and substantially insoluble in thesupercritical fluid.

In the particle formation vessel the substance solution is mixed withthe mixture of supercritical fluid and modifier or with the puresupercritical fluid. In this way the solvent is extracted from thesolution and the substance precipitates as fine particles.

The crucial point of the process for fine particle formation is themixing of the solution with the supercritical fluid: a rapid andintimate mixing causes precipitation of particles and allows a highpowder yield to be obtained.

To have a good mixing, the solution has to be dispersed into thesupercritical fluid in form of small droplets, thus providing a highinterfacial area for mass transfer and a short path for the diffusion ofsupercritical fluid in the solution droplets and preventing the growthof solute particles. In addition, the enhancement of mass transfer ratebetween solution and supercritical fluid allows operation at mildertemperature and pressure conditions. The present invention permits suchoperation.

In addition a high ratio between flow rate of supercritical fluid andflow rate of solution allows the creation of a large excess of thesupercritical fluid over the solution at the moment of their contact,enhancing the driving force for mass transfer of supercritical fluidinto solution and of solvent into supercritical fluid.

As pointed out above, it is necessary to have a good dispersion of thesolution into the supercritical fluid in order to obtain very smalldroplets of solution.

The size of the formed solution droplets is determined by thefluidodynamic conditions in the mixing zone and by the physicalproperties of solution and supercritical solvent, such as viscosity,surface tension, density. These properties are greatly influenced bytemperature and pressure for the supercritical fluid.

The velocity of solution and supercritical fluid at the nozzle outletsis related to the mass flow rate and to the diameter of the outlets.Additionally, it is necessary that the energy pressure of both solutionand supercritical fluid are converted into kinetic energy with a minimumenergy loss.

To get this aim a new nozzle has been designed.

The solution and the supercritical fluid, pure or mixed with themodifier, are introduced in the particle formation vessel in co-currentflow by the nozzle, which provides separate outlets for thesupercritical fluid and the solution. Contact between the solution andthe supercritical fluid first occurs in the particle formation vesseldownstream of the nozzle outlets. This minimises the potential forblockage of the nozzle by the particles which are formed. The respectivedischarges of the supercritical fluid and the solution can expand andmix in the particle formation vessel.

The nozzle has passages for the respective flows comprising a widediameter upstream portion which feeds a narrow diameter downstreamportion. The narrow diameter portion can be short in order to reduce thepressure drop along this portion so that a better conversion of pressureinto kinetic energy is obtained. This overcomes the problems of priorart nozzles which are essentially coaxial tubular arrangements in whicha narrow diameter is maintained along the full length of the nozzle witha significant drop in pressure.

The outlets are preferably located adjacent to each other, for exampleat a centre line spacing of about 3 mm. The outlets are preferablycoplanar.

Preferably the nozzle has one central outlet and a plurality of outeroutlets. The central outlet may serve to carry a flow of solution andthe outer outlets may serve to carry a flow of supercritical fluid. Byproviding a plurality of outer outlets, mixing of supercritical fluidand the solution is promoted. Preferably the outer outlets are arrangedat the same distance from the central outlet. Thus they may be on thesame radius, preferably equiangularly spaced. Again, this assistsmixing.

The outlets may be at the end of separate tubes or the like. It ishowever preferred for the outlets to be provided at downstream ends ofrespective passages through a nozzle body. The passages may for examplebe laser drillings. The nozzle body may be a disk. Thus a preferredarrangement comprises a nozzle in the form of a disk with an outlet atits center and two or more outlets at the same distance from the centerand evenly spaced along a circumference. All the outlets communicatewith the interior of the particle formation vessel. The solution ispreferably introduced into the particle formation vessel through thecentral outlet, while the supercritical fluid, pure or with themodifier, is introduced through the outer outlets.

The passages in the nozzle body have upstream ends which in use are fedwith the supercritical fluid and the solution, respectively. Preferably,the nozzle body is provided with a seal for sealingly separatingrespective upstream ends of the passages therethrough. Thus, the use ofa nozzle body allows the drilling or other formation of the passageswith the ideal dimensions to optimise the fluid flows, whilst thesepassages can be sealed from each other at their upstream ends. In thecase of a central outlet and plural outlets radially outwardly spacedtherefrom, the seal may be annular in form (being e.g. an O-ring) anddisposed radially outwardly of the central outlet and radially inwardlyof the plural outer outlets. A further annular seal is preferablyprovided radially outwardly of the plural outer outlets. Preferably, theor each seal is received in a groove in the nozzle body, e.g. an annulargroove.

The outlets are preferably provided downstream of the apex of conicallytapering portions of the nozzle. The passages may be formed with theseconically tapering portions. Thus a passage may have a relatively widediameter portion at its upstream end, for example 1 mm, followed by aconically tapering portion narrowing to a narrow diameter portion, forexample 20 microns. The narrow diameter portion is referred to herein asan “orifice”. The wide portion and the conical portion may for examplebe mechanically drilled, whilst the narrow portion or orifice may belaser drilled. The length of the wide portion is substantially greaterthan the length of the orifice, so as to allow the nozzle body to berelatively thick in the direction of flow, for example 5 mm, and thuseasy to handle, without causing the orifice to have an excessive length.The length of the wide portion may for example be at least 5 times, morepreferably 10 times, greater than the length of the orifice.

In alternative arrangements, the orifice, with a narrow diameter, mayextend through the full thickness of the nozzle body, but this is notpreferred as the nozzle body would have to be thin (in the direction offlow) and thus difficult to handle.

The expansion of solution and supercritical fluid thus occurs downstreamof orifices. A preferred orifice is characterized by a length todiameter ratio ranging from 5 to 10. It has the advantage over thecapillary of minimizing the pressure energy loss and of efficientlyconverting the pressure energy into kinetic energy.

The nozzle preferably has orifices with diameters ranging from 0.02 to0.1 mm, more preferably from 0.02 to 0.04 mm, and length ranging from0.1 to 0.2 mm. Such dimensions allow very high velocities to be obtainedat the orifice outlet for both solution and supercritical fluid.

In the preferred embodiments, the plural supercritical fluid outlets arepositioned around the solution outlet and at a very short distance(about 3 mm): this configuration allows for the solution to be energizedby the supercritical fluid thus enhancing the dispersion of the solutioninto very fine droplets, providing high interfacial surface between thetwo phases and fast extraction of solvent into supercritical fluid.These phenomena are particularly efficient when the supercritical fluidvelocity at the outlet reaches or is greater than the speed of sound.When the supercritical fluid velocity reaches or is greater than thespeed of sound, a Mach disc is formed which causes the dispersion ofsolution into very fine droplets. This phenomenon is well known and itis widely used in the RESS process (Matson D. W., Fulton J. L., PetersenR. C., Smith R. D., “Rapid expansion of supercritical fluid solutions:solute formation of powders, thin films, and fibers” Ind. Eng. Chem.Res., 1987, 26, 2298–2306).

Even if the supercritical fluid velocity is less, but of the order ofmagnitude of the speed of sound, a substantial enhancement of solutiondispersion is obtained (Subramaniam B., Saim S., Rajewskj R. A., StellaV. Methods for particle micronization and nanonization byrecrystallization from organic solutions sprayed into a compressedantisolvent. U.S. Pat. No. 5,874,029, Feb. 23, 1999).

It is known that during adiabatic expansion of a real fluid through aconvergent-divergent nozzle, the downstream pressure (usually called thecritical pressure) for which the supercritical fluid reaches soundvelocity is related to upstream pressure by the following relation:

$\frac{P_{c}}{P} = \left( \frac{2}{k + 1} \right)^{\frac{k}{k - 1}}$where P is the upstream pressure, P_(c) is the downstream pressure and kis the ratio between c_(p) and c_(v) (specific heat at constant pressureand specific heat at constant volume of the supercritical fluid,respectively). For instance if the supercritical fluid is carbondioxide, for which k=4.81, if the downstream pressure is 10 MPa, theupstream pressure has to be 38.4 MPa to reach the speed of sound i.e. apressure drop of 28.4 MPa are required.

However, with pressure drop of about 4 MPa it is possible to getsupercritical fluid velocity of the order of magnitude of speed of soundfor downstream pressure of 10 Mpa at 40° C.

The speed of sound of a fluid is strongly dependent on pressure andtemperature: the minimum value of speed of sound for carbon dioxide inthe supercritical region is of 208 m/s at 8 MPa and 40° C. To get theadvantage of the above mentioned phenomena it is convenient to workaround these operating conditions when carbon dioxide is used assupercritical fluid.

The preferred nozzle used for the apparatus of the present invention haslaser drilled orifices. The supercritical fluid velocity at the orificeoutlet can be estimated from the energy balance between a section of thesupercritical fluid passage upstream of the orifice (section 1) and asection at the orifice outlet (section 2). The energy balance neglectingthe energy losses can be calculated by the following equation:H ₁+½ρ₁ν₁ ² =H ₂+½ρ₂ν₂ ²

where H₁ and H₂ are the specific enthalpies of supercritical fluidupstream and downstream the orifice respectively; ρ₁ and ρ₂ are thedensities of supercritical fluid upstream and downstream the orificerespectively; ν₁ and ν₂ are the velocities of supercritical fluidupstream and downstream the orifice respectively.

For the production of fine powders from aqueous solutions with the GASprocess using carbon dioxide as supercritical solvent and ethanol asmodifier, it has been found that optimal operative conditions are within8–12 Mpa of pressure and within 35–50° C. of temperature. In theexperimental apparatus used for carrying out the experimental tests thesupercritical fluid mass flow rate was 30 g/min, the solution flow rate0.2 g/min, and the modifier mass flow rate 4 g/min, having set the ratioof supercritical fluid to modifier mass flow rate at 7 and the ratio ofmodifier to solution mass flow rate at 20 and supercritical fluidvelocity at the nozzle outlet at about 300 m/s.

As an alternative to what is described above, the supercritical fluidcan be ethane, ethylene, propane, sulfur hexafluoride, nitrous oxide,chlorotrifluoromethane, monofluoromethane, xenon and their mixtures; thesolvent of the pharmaceutical compound solution can be a supercriticalfluid miscible one such as ethanol, methanol, DMSO, isopropanol,acetone, THF, acetic acid, ethyleneglycol, polyethyleneglycol,N,N-dimethylaniline. The same solvents can be used as modifiers when anaqueous solution of pharmaceutical compound is employed.

DESCRIPTION OF THE DRAWINGS

Certain preferred embodiments of the invention will now be described byway of example and with reference to the drawings, wherein:

FIG. 1 shows a schematic flow sheet of the apparatus used to carry outthe process according to this invention;

FIG. 2 is a schematic section of the nozzle that is used to carry outthe process according to the invention, taken along line A—A of FIG. 3,some parts of the nozzle being shown enlarged in circles;

FIG. 3 is a section of the nozzle on the line B—B of FIG. 2;

FIGS. 4 and 5 are more detailed views similar to FIGS. 2 and 3,respectively;

FIG. 6 is a sectional view of the nozzle arrangement;

FIGS. 7 and 8 are SEM photomicrographs of SIGMA ALP produced under theconditions of example 1;

FIGS. 9, 10 and 11 are SEM photomicrographs of SIGMA lysozyme producedunder the conditions of example 2;

FIGS. 12 and 13 are photomicrographs of trehalose produced under theconditions of Example 3; and

FIG. 14 is a graph showing the particle size distribution of trehaloseproduced under the conditions of example 3.

DETAILED DESCRIPTION OF THE INVENTION

Referring to FIG. 1, the apparatus shown includes a particle formationvessel 22. This is a standard reaction vessel of an appropriate volume.The temperature in the vessel is maintained constant by means of aheating jacket 21. The pressure in the vessel is controlled by means ofa micro metering valve 25.

The temperature and pressure in the particle formation vessel aremeasured by means of a thermocouple 29 and a pressure transducer 30.

The particles formed are retained by a filter 23. This is a stainlesssteel basket, the bottom of which is made by a sintered stainless steeldisk (0.5 micron). A second filter 24 (0.5 micron) is put at the vesseloutlet.

The supercritical fluid is withdrawn from cylinder 3, it is condensed bycooler 4 and pumped by means of pump 8 to the particle formation vesselthrough line 34. Prior to entering into the particle formation vessel,the supercritical fluid is heated to the desired temperature by means ofpre-heater 14 and heater 17. The pre-heater 14 also acts as pulsationdamper. The supercritical fluid is also filtered by means of filter 15(0.5 micron).

Temperature and pressure of the supercritical fluid prior it enters intothe precipitation vessel are measured by means of thermocouple 28 andpressure transducer 43, respectively.

The modifier is withdrawn from tank 2, it is pumped by means of pump 9to line 34 and it is mixed with the supercritical fluid prior to itentering into the particle formation vessel. The modifier is alsofiltered by means of filter 12 (0.5 micron).

Line 34 is equipped with a relief valve 16.

The solution is withdrawn from tank 1, it is pumped by means of pump 10to the particle formation vessel through line 6. The solution is alsofiltered by means of filter 13 (0.5 micron).

In another version of the process the modifier may be introduced intothe particle formation vessel in part with the solution and in part withthe supercritical fluid.

The supercritical fluid, pure or mixed with the modifier, and thesolution are fed into the particle formation vessel 22 by means of anozzle 27.

Downstream of the particle formation vessel 22, the mixture ofsupercritical fluid, modifier and solvent are filtered by means of thefilter 24 (0.5 micron) to retain the particles not previously retainedby filter 23. The mixture of supercritical fluid, modifier and solventis depressurised by means of micro metering valve 25, the supercriticalsolvent is separated from the modifier and the solvent in the separator26, its flow rate is measured by means of mass flow meter 31 and it isdischarged.

FIGS. 2 and 3 show the nozzle that is used to carry out the processaccording to this invention. This nozzle is a distinctive feature of theprocess according to this invention.

The nozzle allows the introduction of the solution and the supercriticalfluid, pure or mixed with the modifier, in the particle formation vesselin co-current flow.

The nozzle provides separate outlets for the supercritical fluid and forthe solution. The nozzle may be made of stainless steel, or of otherappropriate material.

The nozzle 27 has a nozzle body in the form of a disk 36 with an orifice39 at its center and two or more orifices 41 drilled at the samedistance from the center and evenly spaced along a circumference. Theorifices communicate with the interior of the particle formation vessel.The solution is introduced into the particle formation vessel throughthe central orifice, and the supercritical fluid, pure or with themodifier, is introduced into the particle formation vessel through theouter orifices.

The passage 37 for the solution includes a hole of diameter D3. The endof the hole has a conical shape 40. At the apex of the conical end 40there is the laser drilled orifice 39. The length L1 of the centralorifice is 5 to 10 times its diameter D1. The diameter D1 can be chosenin such a way to obtain any desired velocity of the solution at theorifice outlet.

The passages 38 for the supercritical fluid are holes of diameter D4.The end of each hole has a conical shape 42. At the apex of the conicalend 42 there is the laser drilled orifice 41. The length L2 of theorifice is 5 to 10 times its diameter D2. The diameter D2 can be chosenin such a way to obtain any desired velocity of the supercritical fluidat the orifice outlet.

The ratio between length (L1 or L2) and diameter (D1 or D2) of theorifices 39 and 41 are chosen so as to set to a minimum the energy lossand to obtain higher velocities by converting energy pressure intokinetic energy.

In FIGS. 4 and 5 detailed drawings of the nozzle used in the presentinvention are shown. Orifices can be drilled with diameters down to 0.02mm. The nozzles that have been used for carrying out the experimentaltests have orifices of diameter ranging from 0.02 to 0.04 mm.

In another embodiment of the invention, one or more of the outerorifices are drilled in such a way that their axes converge on the axisof the central orifice. The angle formed by the axes of the outerorifices with the axis of the central orifice is comprised between 1 and30°.

The upper surface of the disk 36 of the nozzle 27 is formed with aninner annular groove 50 which extends round the inlet end of the centralpassage 37, and an outer annular groove 52 which extends round the inletends of the passages 38.

FIG. 6 shows the assembly of the nozzle 27. The annular groove 50 of thedisk 36 receives a first O-ring seal 54 and the outer annular groove 52receives a second O-ring seal 56. The disk 36 is received in a cup 58,which also receives a nozzle block 60, the lower end of which is inengagement with the second O-ring seal 56. Over the lower part of itslength the nozzle block 60 is provided with a central lower bore 62which communicates at its upper end with a lateral bore 64. Over theupper part of its length the nozzle block 60 has a central upper bore66. A nozzle shaft 68 extends along the central upper and lower bores66, 62 and has a lower end in engagement with the first O-ring seal 54.The nozzle shaft 68 is formed with a central shaft bore 70. A furtherseal (not shown) would normally be provided around the nozzle shaft 68to seal against the upper part of the nozzle block 60.

In use, liquid solution is fed to the central shaft bore 70 and fromthere to the inlet end of the central passage 37 through the disk 36.The junction between the central shaft bore 70 and the disk 36 is sealedby the first O-ring seal 54. Supercritical fluid, optionally with amodifier, is fed to the lateral bore 64 which communicates with thecentral lower bore 62, and from there to the passages 38 through thedisk 36. The junction between the central lower bore 62 and the passages38 is sealed on the inside by the first O-ring seal 54 and on theoutside by the second O-ring seal 56.

The solution emerges from the central orifice 39 at high velocity and itis broken in fine droplets coming in contact with the supercriticalfluid. The breaking of the solution liquid jet is highly enhanced by thesupercritical fluid emerging from orifices 41, provided that thesupercritical fluid velocity is very high, of the order of magnitude thevelocity of sound at the working temperature and pressure. The effect ofthe supercritical fluid in enhancing the breaking of the solution liquidjet is a crucial one and determines the shape, size and yield of theproduct.

Experimental Procedure

The supercritical fluid is fed to the precipitation vessel by means ofpump 8, which allows setting of the supercritical fluid flow rate. Thetemperature of the supercritical fluid flowing in line 35 is set bymeans of heater 17 to a higher value than the temperature inside theparticle formation vessel, to take into account the temperature loweringdue to the expansion through the nozzle orifices. The modifier is thenadded at a predetermined flow rate to the supercritical fluid by meansof pump 9. The solution is pumped by means of pump 10 into the particleformation vessel when steady state conditions are attained.

After that a certain amount of solution is fed to the particle formationvessel, pumps 9 and 10 are stopped and only the supercritical fluid isfed to the particle formation vessel until the precipitated powder isfree of solvent and modifier.

The particle formation vessel is depressurised and the powder isrecovered.

EXAMPLES

The following examples were carried out using a method according to thepresent invention. The apparatus used is similar to that shown in FIG.1.

Example 1 Preparation of Alkaline Phosphatase (ALP) Particles

In this example, the method of the invention is used to prepare proteinpowders using alkaline phosphatase (ALP).

A solution of ALP (SIGMA Chemicals) in deionized water at aconcentration of 0.2% w/w is used. Carbon dioxide and ethanol are usedas supercritical fluid and as modifier, respectively.

The solution is fed into the particle formation vessel 22 by means pump10 at a flow rate of 0.2 g/min. Supercritical carbon dioxide is fed bymeans pump 8 at a flow rate of 30 g/min, ethanol is fed by means pump 9to line 34 at a flow rate of 4 g/min and it is mixed with supercriticalcarbon dioxide prior to entry into the particle formation vessel.

The supercritical fluid is injected into the particle formation vesselthrough the four external orifices of the nozzle, each with a diameterof 0.04 mm. The solution is injected into the particle formation vesselthrough the central orifice of the nozzle, having a diameter of 0.04 mm.The length of all orifices is 0.2 mm.

Temperature and pressure in particle formation vessel are maintained atT=40° C. and P=10.0 MPa. Precipitated particles are collected on thefilter 23 at the bottom of particle formation vessel, whilesupercritical fluid, modifier and water are collected into cylinder 26at atmospheric pressure.

The solution and the carbon dioxide with the modifier have been fed for240 min, after the solution feed has been stopped, pure carbon dioxidehas been fed into particle formation vessel in order to extract anytrace of solvent and modifier from the precipitated powders. Typically,the particles formation vessel was washed with two volumes of carbondioxide in order to obtain dry powders.

After depressurization, the particle formation vessel is opened and thepowders are recovered.

The yield of the collected powder, was about 70%.

The SEM micrographs (FIGS. 7, 8) show that the obtained powders have anequivalent diameter of less then 1 μm and a narrow size distribution.

The found residual enzymatic activity of ALP was 90%, compared to theunprocessed commercial reagent.

Example 2

Preparation of Lysozyme Particles

In this example, the method of the invention is used to prepare proteinpowders using Lysozyme.

A solution of Lysozyme (SIGMA Chemicals) in deionized water at aconcentration of 0.2% w/w is used. Carbon dioxide and ethanol are usedas supercritical fluid and as modifier, respectively.

The solution is fed into the particle formation vessel 22 by means ofpump 10 at a flow rate of 0.2 g/min. Supercritical carbon dioxide is fedby means of pump 8 at a flow rate of 30 g/min, ethanol is fed by meansof pump 9 to line 34 at a flow rate of 4 g/min and it is mixed withsupercritical carbon dioxide prior to entry into the particle formationvessel.

The supercritical fluid is injected,into the particle formation vesselthrough the four external orifices of the nozzle, each with a diameterof 0.04 mm. The solution is injected into the particle formation vesselthrough the central orifice of the nozzle, having a diameter of 0.04 mm.Length of all orifices is 0.2 mm.

Temperature and pressure in particle formation vessel are maintained at40° C. and 10.0 Mpa respectively.

Precipitated particles are collected on the filter 23 at the bottom ofparticle formation vessel, while supercritical fluid, modifier, waterand solute eventually not precipitated are collected into cylinder 26 atatmospheric pressure.

After that a certain amount of solute is fed into particles formationvessel, pumps 9 and 10 are stopped and only supercritical fluid is fedinto particles formation vessel in order to dry the precipitatedpowders: typically, it needs about two times the volume of the particlesformation vessel to obtain dry powders.

At this point, it is possible to depressurise the particle formationvessel, to open and to recover the powders.

The yield of recovered powder was 90%.

The SEM micrographs (FIGS. 9, 10, 11) show that the obtained powdershave an equivalent diameter of less then 1 μm and a narrow sizedistribution.

The found residual enzymatic activity of ALP was 94%, compared to theunprocessed commercial reagent.

Example 3

Preparation of Trehalose Particles

In this example, the method of the invention is used to preparetrehalose powders from aqueous solutions.

A solution of trehalose dihydrate (SIGMA Chemicals) in deionized waterat a concentration of 2% w/w is used. Carbon dioxide and ethanol areused as supercritical fluid and as modifier, respectively.

The solution is fed into the particle formation vessel 22 by means pump10 at a flow rate of 0.2 g/min. Supercritical carbon dioxide is fed bymeans pump 8 at a flow rate of 30 g/min, ethanol is fed by means pump 9to line 34 at a flow rate of 4 g/min and it is mixed with supercriticalcarbon dioxide prior to entry into the particle formation vessel.

The supercritical fluid is injected into the particle formation vesselthrough the four external orifices of the nozzle, each with a diameterof 0.04 mm. The solution is injected into the particle formation vesselthrough the central orifice of the nozzle, having a diameter of 0.04 mm.Length of all orifices is 0.2 mm.

Temperature and pressure in the particle formation vessel are maintainedat 40° C. and 10.0 Mpa respectively.

Precipitated particles are collected on the filter 23 at the bottom ofparticle formation vessel, while supercritical fluid, modifier, waterand solute eventually not precipitated are collected into cylinder 26 atatmospheric pressure circa.

After that a certain amount of solute is fed into particle formationvessel, pumps 9 and 10 are stopped and only supercritical fluid is fedinto particle formation vessel in order to dry the precipitated powders:typically, it needs about two times the volume of the particlesformation vessel to obtain dry powders.

At this point, it is possible to depressurise the particle formationvessel, to open and to recover the powders.

The yield of recovered powder was 80%.

FIGS. 12 and 13 are SEM micrographs of the obtained powders.

The particle size distribution shown in FIG. 14 has been determinedusing an Aerosizer mo. 3225 (TSI-Amherst) and gives a mean size of 1.89μm.

The invention may be understood in somewhat broader terms. Thus,according to one broad aspect the invention provides apparatus formicron and submicron particle formation of a substance using the GASprocess, comprising a particle formation vessel and means forintroducing a solution of the substance and a supercritical fluid intosaid particle formation vessel, characterized in that said meanscomprise a nozzle having separate outlets for the solution and thesupercritical fluid respectively.

According to another broad aspect the invention provides a nozzle forthe introduction of a solution of a substance and a supercritical fluidin a particle formation vessel for micron and submicron particleformation of said substance using the GAS process, characterized in thatthe nozzle comprises a central outlet to carry a flow of solution and aplurality of outer outlets to carry a flow of pure supercritical fluidor a flow of supercritical fluid mixed with a modifier.

According to a further broad aspect the invention provides a process formicron and submicron particle formation of a substance using the GASprocess, comprising the feeding of a supercritical fluid, pure or mixedwith a modifier, and of a solution, through separate inlets of a nozzle,into a particle formation vessel at controlled pressure and temperature,such that the solvent is extracted from solution by the supercriticalfluid and precipitation of micron and submicron particles occurs.

1. An apparatus for particle formation of a substance using the gasanti-solvent recrystallization (GAS) process, comprising: a particleformation vessel; means for introducing a solution of the substance anda supercritical fluid into said particle formation vessel, said meanscomprising a nozzle having respective passages for the solution and thesupercritical fluid and separate outlets at downstream ends of therespective passages, such that in use contact between the solution andthe supercritical fluid first occurs in the particle formation vesseldownstream of the separate outlets, wherein the passages comprise a widediameter upstream portion which feeds a narrow diameter downstreamportion.
 2. The apparatus as claimed in claim 1, wherein said nozzle hasone central outlet and a plurality of outer outlets, the central outletserving to carry a flow of solution, and the outer outlets serving tocarry a flow of pure supercritical fluid.
 3. The apparatus as claimed inclaim 2, wherein said outer outlets are arranged at the same distancefrom said central outlet.
 4. The apparatus as claimed in claim 1,wherein the nozzle comprises a nozzle body and said respective passagesextend through the nozzle body.
 5. The apparatus as claimed in claim 4,wherein the nozzle body is provided with a seal for sealingly separatingrespective upstream ends of the passages therethrough.
 6. The apparatusas claimed in claim 5, wherein the seal is received in a groove in thenozzle body.
 7. The apparatus as claimed in claim 1, wherein the nozzlehas conically tapering portions, said tapering portions having an apex,and wherein said outlets are provided downstream of the apex of theconically tapering portions.
 8. The apparatus as claimed in claim 1,wherein the outlets are at the downstream ends of orifices, the diameterof said orifices being between 0.02 and 0.1 mm, and a length to diameterratio of said orifices being between 5 and
 10. 9. The apparatus asclaimed in claim 8, wherein the diameter of said orifices is between0.02 and 0.04 mm.
 10. The apparatus as claimed in claim 1, wherein theoutlets are at the downstream ends of orifices drilled in such a waythat their axes converge, the angle formed between the axes beingbetween 1 and 30°.
 11. The apparatus as claimed in claim 1, farthercomprising means for introducing a modifier in said particle formationvessel through said nozzle.
 12. The apparatus as claimed in claim 1,wherein a respective outlet carries a flow of the solution mixed with amodifier.
 13. The apparatus as claimed in claim 1, wherein a respectiveoutlet carries a flow of the supercritical fluid mixed with a modifier.14. A nozzle for particle formation of a substance in a particleformation vessel using the gas anti-solvent recrystallization (GAS)process, the nozzle being for the introduction of a solution of saidsubstance and a supercritical fluid in the particle formation vessel forparticle formation of said substance, the nozzle comprising: respectivepassages for the solution and the supercritical fluid and separateoutlets at downstream ends of the respective passages, such that in usecontact between the solution and the supercritical fluid first occursdownstream of the separate outlets, wherein the passages comprise a widediameter upstream portion which feeds a narrow diameter downstreamportion.
 15. The nozzle as claimed in claim 14, comprising a centraloutlet to carry a flow of solution and a plurality of outer outlets tocarry a flow of pure supercritical fluid or a flow of supercriticalfluid mixed wit a modifier.
 16. A process for particle formation of asubstance using the gas anti-solvent recrystallization (GAS) process,comprising: the feeding of a supercritical fluid, pure or mixed with amodifier, and of a solution of the substance wherein said solutioncomprises a solvent, through a nozzle, into a particle formation vesselat controlled pressure and temperature, such that the solvent isextracted from solution by the supercritical fluid and precipitation ofparticles of the substance occurs, wherein the supercritical fluid andthe solution are fed through respective passages of the nozzle to exittherefrom via separate outlets at downstream ends of the respectivepassages, with contact between the supercritical fluid and the solutionfirst occurring in the particle formation vessel downstream of theseparate outlet, and wherein the passages comprise a wide diameterupstream portion which feeds a narrow diameter downstream portion. 17.The process as claimed in claim 16, wherein said solution is introducedinto the particle formation vessel mixed with the modifier.
 18. Theprocess as claimed in claim 16, wherein said supercritical fluid isselected from the group consisting of ethane, ethylene, propane, sulfurhexafluoride, nitrous oxide, chlorotrifluoromethane, monofluoromethane,xenon and their mixtures.
 19. The process as claimed in claim 16,wherein said modifier is selected from the group consisting of ethanol,methanol, DMSO (Dimethyl Sulfoxide), isopropanol, acetone, THF(Tetrahydrofuran), acetic acid, ethyleneglycol, polyethylencglycol, andN,N-dimethylaniline.
 20. The process as claimed in claim 16, wherein thesolution is an aqueous solution and contains the substance, thesubstance comprises a compound, the supercritical fluid is carbondioxide and the modifier is ethanol.
 21. The process as claimed in claim20, wherein the pressure in the particle formation vessel is between thecritical pressure of carbon dioxide and 30 MPa and the temperature inthe particle formation vessel is between 30 and 80° C.
 22. The processas claimed in claim 21, wherein the ratio between the mass flow rate ofcarbon dioxide and modifier is between 2 and 40 and the ratio betweenthe mass flow rate of modifier and of aqueous solution is between 5 and40.
 23. The process as claimed in claim 21, wherein the criticalpressure is between 8 and 12 MPa and the temperature of the particleformation vessel is between 40° and 50° C.
 24. The process as claimed inclaim 22, wherein the carbon dioxide velocity at the respective nozzleoutlet is of the order of magnitude of the speed of sound in the carbondioxide at the temperature and pressure in the particle formationvessel.
 25. The process as claimed in claim 22, wherein the ratiobetween the mass flow rate of the carbon dioxide and modifier is between6 and 8 and the ratio between the mass flow rate of modifier and ofaqueous solution is between 10 and 25.